The invention relates to the fields of polymer chemistry and nucleic acid transfection.
There are currently two methods used to silence the expression of a target gene: antisense oligonucleotides (ODN) and RNA interference (RNAi). Until recently, ODNs had been the main hope for decreasing or silencing gene expression for therapeutic purposes. ODNs are short pieces of DNA or RNA complementary to messenger RNA (mRNA) sequences. They function by hybridizing with the mRNA to create a double stranded region. In the case of DNA ODNs, a RNA-DNA duplex is formed, which can be recognized by an RNAse, thus degrading the mRNA and eliminating protein expression by the targeted sequence. This process has been successful in vitro, but the lack of an appropriate delivery mechanism has limited in vivo applications [9]. The second method of gene silencing is RNAi, first described in worms in 1998 [3]. It was later found that this phenomenon also operates in insects [4], frogs [5], mice [6], and it is now believed to exist in all animals. The natural function of RNAi appears to be protection of the genome against invasion by mobile genetic material elements such as transposons and viruses. Transposons and viruses produce aberrant RNA or dsRNA when they become active [7], and it is, thus, desirable to eliminate such nucleic acids. Once this dsRNA (>30 nucleotides (nt)) is inside the cell, the process of RNAi begins with the enzymatic degradation of the dsRNA into roughly 22 nucleotide-long dsRNA segments, termed small interfering RNA (siRNA) [8]. The siRNA along with nucleases and other proteins assemble into the RNA induced silencing complex (RISC). The RISC enables the antisense strand of the siRNA to bind to the complementary sequence of mRNA, and, this bound complex, in turn, induces the degradation of the complementary mRNA. In mammalian cells, if the process is initiated by long dsRNA (>30 nt), the interferon response is activated and protein kinase R and RNAse I are activated leading to the arrest of all protein synthesis. Apoptosis of the cell following delivery of siRNA (<30 nt) has been shown to be an efficient way of silencing a specific gene without compromising gene expression, more generally, in mammalian cells. The structure of siRNA typically consists of 21-22 nucleotide double stranded RNA (dsRNA) with 2-3 nucleotide long 3′ overhangs [10]. Silencing genes via siRNA is a highly efficient process, reported to be 1000-fold more effective than ODNs [9]. The delivery of siRNA for biomedical applications is very promising because of its stringent sequence-specific action. A single base mismatch over the length of the siRNA is enough to prevent induction of the response [10].
The ability to down-regulate gene expression specifically using the RNAi pathway or antisense oligonucleotides and to up-regulate gene expression via non-viral gene delivery has tremendous potential as a therapeutic and as a tool in basic science. However, an efficient delivery system capable of specifically delivering small interfering RNA (siRNA), antisense oligonucleotides (ODN), or plasmid DNA (pDNA) to target cells is currently unavailable. Cationic polymers that can self assemble with nucleic acids based on charge are some of the most widely studied and commonly utilized gene delivery vehicles. Limitations of cationic-based gene delivery systems include toxicity, aggregation, and unpacking of the DNA. siRNA delivery suffers from many of the same limitations as non-viral gene delivery approaches, including targeted internalization and endosomal escape, for which a delivery system is yet to be found that is as efficient as viral vectors. Following receptor-mediated uptake into coated pits and endocytosis, the endosomes are shuttled toward lysosomal fusion, with degradation of their contents. Only a small fraction of the endosomal contents escape.
siRNA has been shown to be functional in vitro and in vivo, mediating targeted gene silencing in a variety of models. Reporter genes have been extensively utilized as proof of principle for siRNA delivery both in in vitro systems [9, 13] and in adult mice [1, 21]. The silencing of therapeutically relevant genes has also shown some success in the inhibition of neovascularization by delivering siRNA targeting for VEGF [21].
Silencing of genes using siRNA requires that the siRNA be internalized by the cell and transferred to the cytosol where the RNAi pathway is activated, the targeted mRNA destroyed, and the gene of interest silenced. siRNA delivery suffers from many of the same limitations as other non-viral gene delivery approaches such as targeted internalization and endosomal escape, for which a delivery system is yet to be found that is as efficient as viral vectors. Nevertheless current strategies for the delivery of siRNA employ the same delivery vectors as used for non-viral gene delivery such as cationic polymers, lipids [11, 12], and peptides [13], which self assemble with siRNA electrostatically. Two key differences in the mechanism of action of siRNA and plasmids are that siRNA does not need to cross the nuclear barrier to be active and that siRNA is much smaller in size with the possibility of chemical modifications without loss of activity [11]. The fact that siRNA does not need to enter the nucleus is a marked advantage since efficient delivery of DNA to cells requires actively dividing cells where the nuclear membrane is compromised or with vectors that contain nuclear localization sequences. Furthermore, since siRNA is double stranded, it is less susceptible to degradation than other RNA silencing approaches making it a more robust material for chemical modification and delivery. The cationic polymer delivery system for plasmid DNA has been extensively modified with functional domains to design a synthetic vector capable of overcoming the barriers to gene transfer and may be applied to siRNA delivery.
The ability to silence any gene in the genome can be used to augment healthy tissue formation and wound healing by down regulating key molecules, which when expressed, suppress instead of induce tissue formation or regeneration. For example, after abdominopelvic surgery, the accumulation of fibrin between adjacent sides of the wound prevents the regeneration of healthy functional tissue. Abdominal surgery near organs, such as fallopian tubes or the small intestine, poses particular problems since adhesions can constrict the tubes and cause infertility [2, 3], In 1994, adhesions occurred in 90 percent of the 3.1 million US patients undergoing abdominal surgery, with approximately 15 percent undergoing secondary procedures to remove adhesions [4]. Although barriers and polymer lavages have been introduced, the problem remains largely unsuccessfully treated. As to mechanism, postoperative adhesion formation has been associated with a decreased capacity of the mesothelial cells to degrade intra-abdominal deposited fibrin as a result of inhibition of plasminogen activator activity (PAA) by PAI-1 [5]. PAI-1 activity is regulated in part by HIF-1α, a transcription factor, which is, in turn, upregulated by hypoxia in the site of injury [6]. Therefore, targeting the mRNAs of PAI-1 and HIF-1α could be used to tip the balance between fibrin formation and fibrin resorption toward overall resorption, thus preventing abdominal adhesions. HIF-1α regulates the production of a number of other scar-forming proteins, including transforming growth factor β (TGFβ), and, as such, inhibition of the transcription factor may have multiple positive effects. It is also noteworthy that it has been previously been found that treatment of surgical subjects with tissue plasminogen activator and ancrod [7,8] did not inhibit favorable healing, demonstrating that the coagulation/fibrinolysis balance within the tissue is more robust than in the peritoneal cavity. Given that PAI-1 and HIF-1α are produced by mesothelial cells on the surface of the peritoneum and fibroblast invading the fibrin coagulum at the site of injury, multiple cellular targets are present to which to deliver such therapeutics.
Thus, there is a need for new delivery systems for nucleic acids and other negatively charged molecules and for effective treatments for diseases and conditions, such as surgical adhesions.